Rapid methods for assay of enzyme substrates and metabolites

ABSTRACT

Method and apparatus for quantitating the amounts of enzyme substrates and metabolites present in biological fluids using spot test techniques. The tests utilize both fixed, preselected color standards and substrate and metabolite standards freezedried on transparent membranes or in porous pads for comparison. Other reagents including enzymes, dyes, cofactors are also freeze-dried in absorbent pads. The assembly may utilize any or all of the following in various combinations: porous discs of materials such as glass fibre filters to store reagents, to serve as liquid reaction volume measurement, and to assist in removing cells from blood samples; microporous membranes to act as barriers to blood cells; a protein enrichment membrane or a dialysis membrane to restrict passage of large molecules to the indicator zone; and a transparent window which may be a second microporous membrane or impermeable plastic on which indicator dyes are dried. In assembled format, the test plates, slides or discs fulfill all requirements for conducting the selected test: no added instrumentation, controls or other measurements are required. These test devices may assist in the diagnosis of a number of pathological conditions which give rise to abnormal levels of metabolites or enzyme substrates. Methods for rapid assay of serum cholestrol, uric acid, testosterone, androsterone and galactose have been indicated as examples. A wide variety of other compounds found in biological fluids may also be assayed by similar technology.

Write States atent [191 Moyer et a1.

1*Feb. 12, 1974 RAPID METHODS FOR ASSAY OF ENZYME SUBSTRATES ANDMETABOLITES [75] lnventorsz Rudolph H. Moyer, W. Covina;

Donald J. Sibbett, Cucamonga, both of Calif.

[73] Assignee: Geomet,lncorporated, Rockville,

[ Notice: The portion of the term of this patent subsequent to Apr' 16,1989, has been disclaimed.

[22] Filed: Feb. 25, 1971 [21] Appl. No.: 118,903

[52] US. Cl. 195/127, 195/1035 R, 23/253 TP [51] Int. Cl Cl2k 31/14 [58]Field of Search... 195/103.5 R, 127; 23/253 TP 5/1972 Moyer et al195/1035 R Primary Examiner--Alvin E. Tanenholtz Assistant Examiner-MaxD. Hensley [57] ABSTRACT Method and apparatus for quantitating theamounts of enzyme substrates and metabolites present in biologicalfluids using spot test techniques. The tests utilize both fixed,preselected color standards and substrate and metabolite standardsfreeze-dried on transparent membranes or in porous pads for comparison.Other reagents including enzymes, dyes, cofactors are also freeze-driedin absorbent pads. The assembly may utilize any or all of the followingin various combinations: porous discs of materials such as glass fibrefilters to store reagents, to serve as liquid reaction volumemeasurement, and to assist in removing cells from blood samples;microporous membranes to act as barriers to blood cells; a proteinenrichment mem- 'brane or a dialysis membrane to restrict passage oflarge molecules to the indicator zone; and a transparent window whichmay be a second microporous membrane or impermeable plastic on whichindicator dyes are dried. ln assembled format, the test plates, slidesor discs fulfill all requirements for conducting the selected test: noadded instrumentation, controls or other measurements are required.

These test devices may assist in the diagnosis of a number ofpathological conditions which give rise to abnormal levels ofmetabolites or enzyme substrates. Methods for rapid assay of serumcholestrol, uric acid, testosterone, androsterone and galactose havebeen indicated as examples. A wide variety of other compounds found inbiological fluids may also be assayed by similar technology.

11 Claims, 9 Drawing Figures PATENTEUFEB 1 2 3.791 .933

INVENTURH RUDOLPH H. MOYER DONALD J. SIBBETT ATTORNEYS PATENIEB HEB!21914 SHEEI 2 BF 2 mvmmons RUDOLPH H. MOYER DONALD J. SIBBETT ATTORN EYRAPID METHODS FOR ASSAY OF ENZYME SUBSTRATES AND METABOLITES The presentinvention relates to extensions and improvements of the concepts andprinciples disclosed in our copending applications, Ser. No. 63,842,entitled Method and Apparatus for Quantitating Enzyme Activity now US.Pat. No. 3,663,374, filed Aug. 14, i970, and Ser. No. 110,185, entitledImproved Methods and Apparatus for Assaying Enzyme Activity, filed Jan.27, 1971.

BACKGROUND OF THE INVENTION The human body is composed of many differentorgans and several different types of tissues with characteristicfunctions. Most of these tissues contain relatively large amounts ofsecondary specialized materials necessitated by the nature and functionof the tissue. Examples of these specialized compounds include:

1. phospholipids, cholesterol and glycolipids in brain and nerve tissue,

2. glycogen in liver,

3. hemoglobin in red blood cells,

4. hormones in endocrine glands,

5. myosin in muscle, etc.

Both the state of nutrition or pathological conditions markedlyinfluence the composition of the body tissues. Such variations arefrequently reflected in the composition of the body fluids, particularlyblood, which forms the basis for much of modern clinical diagnosis. Afew examples of typical metabolic compounds of interest and diseasesassociated with them are found in the following table:

Increased in diabetes mellitus, starvation, von Gierke's disease,eclampsia, selected nervous disorders, etc.

Increased after ether anesthesia; Decreased in scurvy.

Increased by dehydration;

Decreased in chronic hepatitis, hepatic cirrhosis, anemias, manydiseases.

Increased in acute hepatitis, various jaundices, selected anemias, acuteyellow .atrophy, malaria hemolytic septicemia, incompatible blooodtransfusions, etc. Increased in pheochromatoma, extra medullarychromaffintumors.

Increased levels in arteriolar sclerosis, pancreatitis, diabetesmellitus, nephrotic syndrone, hypophysectomy, multiple sclerosis,leukemia, eclampsia, etc.; Decreased levels in anemia, acute infections,hyperthroidism, epilepsy and hepatic diseases.

Increased in muscular dystrophy, myastenia gravis, hepatic carcinoma,diabetes. "lncreasdTridiabetes. biieimbhia. nephritis,

urinary obstructions, intestinal obstructions, tetany;

Decreased in amyotonia congenita, anemia, leukemia, advanced nephritismuscular atrophy.

Increased in galactosemia, some liver diseases.

Increased in diabetes mellitus, hyperpituitarism, coronary thrombosis,increased intercranial pressure, shock, infarctions, hyperthyroidism,chronic nephritis, urinary obstructions;

Decreased values in hyperinsulinism, hepatic insufficiency, pancreaticadenoma, Addison's disease, hypothyroidism, etc.

Present with gastric carcinoma, chronic gastritis, dilatation of thestomach, pyloric obstruction, etc.

Acetone (urine) Ascorbic Acid (blood and urine) Albumin Bilirubin(blood) Catechol amines Cholesterol Creatine Creatinine G alactose 6OGlucose Lactic Acid (gastric) COMPOUND COMPOSITION CHANGE AND DISEASEPyruvic Acid (blood) Increased in vitamin B and thiamine insufficiency,fever, carcinoma, cirrhosis of the liver, von Gierke's disease,hyperthyroidism, cardiac decompensation. Increased in renal impairment,intestinal obstruction, dehydration, pneumonia, bleeding peptic andduodenal ulcers, etc; Decreased in acute hepatic insufficiency,nephrosis, chronic wasting diseases, amyloidosis, pregnancy.

Increased in various renal insufficiency diseases: acute and chronicnephritis, urinary obstructions, metallic poisoning such as by mercuryand lead, hypertension; metabolic disturbances: gout, diabetes withacidosis: after exercise, rapid destruction of cell nuclei: leukemia,polycythemia, malignant tumors, pernicious anemia, etc;

Decreased in pernicious anemia (relapse), acute yellow atrophy of theliver, salicylate and atophan therapy.

Urea Nitrogen Uric Acid Assays of metabolites and/or enzyme substratesin body fluids have been used for a considerable period as an importantaid in diagnosis of pathological conditions in humans. Rapid assays ofmost of these compounds have not been possible, particularly withoutinstrumentation and trained personnel. Utilization of modifications ofthe techniques described in the aforesaid copending applications forpatents makes available rapid and simple tests for many of theseindicators of physiological disorders.

SUMMARY OF THE INVENTION The methods and apparatus utilize simple butselective spot test procedures for assay of components of the blood,urine, spinal fluid, etc. For measurements which involve inhibition ofenzyme reactions, indicator color development on test spots is comparedto color development on reference spots. In such cases, reference spotscontain standardized amounts of enzymes and/or inhibitory agents. Forsubstrates or metabolites in biological fluids where measurement orassay is dependent only on conversion of the compounds of interest,direct comparison between the color developed on the test spot and acolor chart calibrated in terms of concentration may be utilized.

Illustrative embodiments of apparatus and working embodiments ofpractical methods of the invention will be explained with reference tothe accompanying drawings in which:

FIG. 1 is a plan view of a format suitable for comparison between rateof color development on standard spots and on a test spot;

FIG. 2 is an end elevational view of the structure of FIG. 1;

FIG. 3 is a fragmentary exploded view of a stacked or assembled array ofthe components of a test spot of FIG. 1;

FIG. 4 is a plan view, a portion being broken away for clarity, of aformat for determining final color development indicative of progress ofthe substrate-limited process employed as a basis for analysis;

FIG. 5 is an end elevational view of the embodiment of FIG. 4;

FIG. 6 is a plan view showing a format similar to that of FIG. 4 butdisclosing a different form of readout means;

FIG. 7 is an end elevational view of the embodiment of FIG. 6;

FIG. 8 is a plan view of a further format adapted for use with fixedcolor standards incorporating an immobile color index; and

FIG. 9 is an end elevational view of the embodiment of FIG. 8.

Referring now in more detail to the drawings, the format of FIG. 1 isdesigned for the type of analysis which requires comparison between therate of color development on standard spots and that on a test spot.This arrangement is particularly appropriate for analysis involvingenzyme reaction inhibition such as exemplified by a cholesterol assaysuch as indicated hereinafter. The test plate is generally designated 10and includes thereon a plurality of test spot areas A, B, C, D and E.Spots A, B, and C contain standards, as will appear hereinafter,representing normal, elevated and very high enzyme levels respectively.Spot D is a blank and spot E is for assay of the test specimen andcontains all reagents. A reordering of spots can be appropriate asdesired.

The test plate 10 consists of two slides, 14, 16 of appropriatedimensions and preferably are of high impact, opaque polystyrene sheets.The sheets or slides, as assembled, are fastened by corner eyelets 18and plastic spring clamps 20 are used to cover the edges of theassembly. The spring clamps maintain effective seals between various ofthe components, such as the retaining rings and absorbent glass fibrediscs on which required reagents are impregnated. The spot test formatcomponents and arrangement are shown in FIG. 3. Item 22 is ahigh-impact, circular polystyrene retaining ring of one-fourth inchdiameter with a one-eighth inch hole in its center. This ring which maybe made from clear plastic of 0.015 inch thickness is sealed to theupper side of an opaque, white, high-impact polystyrene slide 14measuring 1 inches X 3 A inches. A 0.161 inch diameter hole 24 in theupper slide 14 is filled with two discs of glass fibre padding (WhatmanGF/A glass fiber paper has been successfully used,) which are designatedas Items 26 and 28. These absorbent discs are retained in the holeduring preparation of the assembly by sealing membranes 30 such asmicroporous filters onto the bottom side of the upper slide. For assayof metabolites and substrates, materials such as Metricel P.E.M.(average pore diameter 0.0075 microns), Acropor AN and Ion ExchangeAcropor, all marketed by the Gelman Instrument Company (Ann Arbor,Michigan), have been found useful and appropriate. Metricel PEM is usedto concentrate protein molecules on the upper side of the barrier.Acropor AN, may be used to filter out blood platelets and cells. AcroporIon-Exchange filters may be used to remove interfering anions andcations from contact with the dyes which are deposited on the uppersurface ofa window 32. Another absorbent disc 34 is placed between themembrane 30 and the window 32. Its function is to assist in drawingliquid through the membrane 30, to serve as reaction volume, as do discs26 and 28, and to distribute the reacting liquid mixture uniformlyacross the viewing window 32. The viewing window 32 may be utilized asthe reservoir for indicator dyes and enzymes. These are deposited insolution and freeze-dried on its upper surface. When the liquid reactionmixture diffuses through the assembly associated with the upper slide,color is developed at the interface between Items 34 and 32. The bottomslide may be sealed to another ring 36 such as the upper retainer 22.The window, 32

may be either a clear impermeable plastic or a transparent film such asNuclepore (General Electric Co.) sealed across the lower ring 36. By useof the permeable film, it has been found possible to avoid the formationof bubbles at the interface between members 34 and 32. Bubbles in thisposition may make color development uneven and difficult to accuratelycompare. The lower ring and the plactic spring edge binders and cornereyelets 18 serve to maintain the integrity of the liquid seal in allparts of each single spot assembly. The two plates are sealed togetherin a dry atmosphere 10 percent relative humidity) and stored in gastight envelopes.

Another example of a test plate format which may be utilized where therate of color development is not of concern, is shown in FIG. 4. Thistype of test plate is employed effectively for most determinations ofmetabolites and enzyme substrates. In such determinations completeconversion of the metabolite by enzymes is usually involved. The finalcolor development is indicative of the progress of the substrate-limitedprocess which has been employed as the basis for analysis. Thisembodiment includes a test plate 38 which is similar in size andconstruction materials to that in FIG. 38. However, only spot sites forthe actual test F and blank G are required. The test spots are assembledas shown in FIG. 3. A color slide 40 which is calibrated in terms ofmetabolite concentrations corresponding to equilibrium development ofcolor is operatively engaged with plate 30. The shaded portion 42represents the colored section. It is graduated in terms of thechromogen utilized. For example, for analyses in which the nitro bluetetrazoleum is used, the color slide varies from white to dark blue. Theread-out side 44 of the color slide is calibrated in terms of theconcentration of the metabolite. For the uric acid determinationindicated below brown colors corresponding to normal values (2-6 mgpercent in serum) have been placed in the center of the slide. Darkerbrown colors (O-dianisidine is the chromogen in this determination) onthe right hand side are indicative of uric acid concentrations above thenormal range. Lighter browns on the left hand side of the slidecorrespond to the color developed by sub-normal uric acid levels. Serumconcentrations which correspond to these levels have been indicated onthe upper section of the color slide. A logarithmic scale has beenutilized.

In practice, the determination is made as follows:

1. Liquid samples are placed on the bottom of the slide, (Item 22 inFIG. 1).

2. The slide is inverted as color development progresses.

3. At approximately 5 minutes after step I, the color on the test sideis matched with that on the color slide by moving the slide until thebest possible color match is achieved in the center of window 46.

The analysis is achieved by reading off the numerical value of themetabolite concentration opposite the index mark at the center of thewindow. The units of the concentrations are printed on the right handend of the slide. Comparison of the color match at later reaction timesmay be used to confirm the analysis.

The test format shown in FIG. 6 is similar to that in FIG. 4. However, arotating color wheel 48 has been employed in the place of the slide.Measurements are achieved by rotation of the wheel and read out on theinner numerical scale 50 opposite the index. In FIG. 6 the indicateditems are as follows:

Item H is the site utilized for sample testing;

Item I is the blank;

Item 52 is the colored portion of the wheel 48 which varies fromcolorless (white) to a color corresponding to excessively high values ofthe assayed metabolite;

Item 54 is the window used to isolate a portion of the color wheelcorresponding to a match with the test site, H;

Item 50 shows the scale calibrations in terms of metaboliteconcentrations; and

Index 56 indicates the metabolite concentration corresponding to thematched color.

Another form of the comparator may utilize a fixed color strip andcalibration printed on the surface of the test plate. However, since theeye views all densities of the chromogen color simultaneously, theaccuracy of the assay is decreased. Such a method is shown in FIG. 8.

In FIG. 8, the blank and test sites are shown at J and K. The colorscale, 58, varies from colorless to excessively intense for thechromogen and assay of interest. Concentration scale 60 corresponds tothe color levels on 58.

A number of examples are indicated below to show the application ofthese techniques.

EXAMPLE 1: BLOOD URIC ACID Variation of uric acid in the blood from thenormal range of 2.0 to 5.0 mg percent accompanies a wide range ofmetabolic, blood, renal and liver diseases.

Analysis utilized the following method: In the presence of the enzymeuricase, uric acid is convertible to allantoin and hydrogen peroxide.The formation of hydrogen peroxide may be detected by the oxidation of anumber of chromogens in the presence of peroxidase. Methods utilizingthis technique have been examined by P. l-Ioworth, and J. Zilva, J.Clin. Path., 21, 192 (1968); G. Domagk and H. Schilicke, Anal. Biochem.22, 219 (1968), and C. Nordschow, Anal. Biochem. 31, 6 (1969).schematically, the reactions involved are:

Uric Acid H O Allantoin H 0 CO H 0 O-dianisidine 2251222., brown pigmentH where O-dianisidine is the chromogenic election donor. Dyes such as2,6-dichlorophenol-indophenol may also be employed.

Procedures and reagents utilized to impregnate the absorbent glass fiberpads of the test and blank spot which are assembled in a configurationsuch as indicated in FIG. 3 are:

1. For use with whole blood samples, glass fiber pads 26, 28, were (a)washed with glacial acetic acid, (b) washed with glass distilled water,(c) dried, (d) saturated with 20 p. l of a solution of bovine serumalbumin containing mg/ml and (e) dried under vacuum in a desiccator.

2. Glycine borate buffer: 5.0g glycine, 3.8 lg sodium tetraborate and42mg disodium salt of ethylene diamine tetracetic acid were dissolved in80 cc of water. The pH was adjusted to 8.50 by the addition of 2Nhydrochloric acid. The solution was made up to 100 ml and used as thebuffering solution.

3. Uricase: Worthington uricase powder was dissolved in 0.5 ml of [/10diluted buffer so as to have approximately 1 IU/ml. Pads 26 and 28 weresaturated with 20p.l and freeze-dried.

4. O-Dianisidine Hydrochloride-Peroxidase Mixture:

A solution containing 3.5 mg'of O-dianisidine hydrochloride(recrystallized) and 0.1 mg horseradish peroxidase in 2.5 ml wasutilized to deposit the chromogen on the viewing surface 32. Thesolution also contained 3.0 mg/ml dextran (Dextran, Clinical, NutrionalBiochemicals Corp.). A volume of Sul was freeze-dried on the transparentNuclepore membrane which was sealed to the ring 36.

In this analysis use of the GELMAN PEM filter as member 30 isappropriate. Readout utilizes the plate format shown in FIGS. 4, 6, or8.

EXAMPLE 2: TESTOSTERONE AND ANDROSTERONE Low levels of the male sexhormones may be involved in or indicative of disorders such as adrenalinsufficiency, hypogonadism, hypopituitarism, osteoporosis andrefractory anemia. High levels may be indicative of adrenalhyperactivity or ovarian tumor. In the adult male, blood levels oftestosterone are of the order of 0.5ug percent. Urine samples may alsobe used for assay.

Testosterone and androsterone may be determined by utilizing the sameprocedure but with different enzyme reagents. The determination oftestosterone may be carried out with Worthington HydroxysteroidDehydrogenase (Code: STDI-I); androsterone requires WorthingtonHydroxysteroid Dehydrogenase (mutant) (Code: STDHM), The method utilizeddepends upon the formation of reduced nicotinamideadenine dinucleotide(NAD.H) from the reaction of the hormone with the appropriate enzyme.The product NADH may be used to develop the color of nitro blue formazanfrom nitro blue tetrazoleum (NBT) utilizing N-methyl phenazoniummethosulfact (PMS) as an electron carrier. schematically the method fortestosterone consists of the following:

Hydroxysteroid I. Testosterone-l-NAD Dehydrogenase (Code STDH) NADH A-androstene-3, 17 -di0ne whereNAD is nicotinanide adenine dinucleotide.

2. NAD.I-I PMS-- NAD PMS.H PMS is N-methyl penazonium methosulfate.

3. PMS.H NBT" PMS Nitro blue formazan (dark blue) PMS.H is the reducedform of PMS.

The initial step has been described by H. Carstensen Nature 212, 1604-5(1966). Detection of NADH by the reaction with PMS and nitro bluetetrazoleum has been described by these inventors in previousbiochemical spot tests.

An indicated in Example 1, the glass fiber pads 26, 28 and 34 werepretreated with purified human serum albumin. After drying they wereimpregnated with 30p.l of the following solution made up in 0.03 molarpyrophosphate buffer (pH 8.8):

REAGENTS CONCENTRATIONS (mg/ml) Nicotinamide adenine 0.8-1.2

dinucleotide (NAD) 50-200 Talahay units REAGENTS HydroxysteroidDehydrogenase N-Methly phcnazonium metho sulfate Marcus. P l andTalahay. P.. J. Biol. Chem. 218. 66! (I956) The transparent layer 32 wasimpregnated with 5 l of:

REAGENTS CONCENTRATIONS (mg/ml) Nitro Blue tetrazoleum Dextran(Dextran,Clinical 200,000-300,000, Nutritional Biochemicals Corp.)

The plate format of FIGS. 5 and 6 are most appropriate. After additionof blood samples to the upper side of the test plate, the reaction isallowed to proceed for approximately 5 minutes at room temperature.Devel- EXAMPLE 3: GALACTOSE Galactosemia is a disorder of galactosemetabolism which if untreated results in cirrhosis of the liver,blindness and mental retardation. It is caused by the absence ofgalactose-l-phosphate uridyl transferase, an occasional hereditarydefect. Liver damage may also give rise to increased levels of galactosein the urine and blood. Under normal conditions, galactose is absorbedrapidly from the intestines and blood and urine levels are negligible.The assay may also be utilized to test carbohydrate metabolism followinggalactose inges-' tion.

The schematic procedure with blood samples which has been indicated byH. Roth, S. Segal and D. Bertoldi, Anal. Biochem., 10, 32 (1965) and .I.M. Sempere, C. Gancedo and C. Asensio, Anal. Biochem., 12, 509 (1965) isas follows:

1. D-galactose 02% D-galactohexodialdose +H O 2. H O-dianisidine brownpigment H O As indicated in Example 1, above, the glass fiber pads arecoated with human serum albumin. The pads 26, 28, and 34, areimpregnated with 30;}.1 of the following; reagent solution made up in0.01M phosphate buffer (pH 7.0):

REAGENT CONCENTRATION (mg/ml) Galactose Oxidase Approximately 0.02

(Worthington, Code GAO) The pads were freeze-dried as indicated in thepreceeding discussion.

The peroxidase-O-dianisidine reagent is freeze-dried on the window ortransparent membrane 32 utilizing a volume of 5p.l. The indicatorsolution is comprised of the following reagents in 0.01 M phosphatebuffer (pH 7.0):

REAGENT O-dianisidine (Recryslallized) Peroxidase (WorthingtonPeroxidase D, Code HPOD) Dextran (Dextran. Nutritional BiochemicalsCorporation) CONCENTRATION (mg/ml) 02 approximately 0.l

Assembly and storage techniques are indicated above.

EXAMPLE 4: CHOLESTEROL Abnormal cholesterol levels in the blood may beused to indicate many disorders involving lipid metabolism. (Table 1indicates a selection of such diseases). Normal values of serumcholesterol vary between 180-260 mg percent for women and -250 mgpercent for men.

Rapid assay of serum cholesterol may be achieved utilizing the spot testformat of this invention in conjunction with the relatively specificinhibition of the enzyme ,B-glucuronidase by cholesterol. This reactionhas been studied by A. L. Tappel and C. J. Dillard, J. Biol. Chem. 242,2463-69 (1967). The methods utilized involved measurement of the amountof phenolphthalein released during the hydrolysis of phenolphthaleinglucuronide by B-glucuronidase.

1. Phenolpthalein glucuronide H phenolphthalein D-glucuronic acid 2.Phenolpthalein base pink-red color (anion) The cholesterol screeningassay utilized the format indicated in FIG. 1. In its preparation, theabsorbent glass fiber pads are treated with serum albumin as previouslydescribed. The two upper pads 26 and 28 are impregnated with 0.20p.l ofan enzyme solution containing 2.0-6.0 mg of B-glucuronidase(Worthington,

Bovine Liver, Code GL) in 0.1M acetate buffer (pH 4.5). The upperassembly is then freeze-dried. The glass fiber pad below the membranefilter 30 is impregnated with a substrate solution containing 8mg/ml ofphenolphthalein glucuronide (Sigma) in 0.1M acetate buffer. This pad isfreeze-dried separately.

Two methods may be utilized for obtaining the anionic red color from thereleased phenolphthalein:

l. A transparent membrane 32 may be utilized which is moistened after anappropriate reaction interval (5-10 minutes) with a basic solution sucha 0.2M glycine buffer (pH 10.4), or,

2. A thin layer of a precipitated base such as calcium carbonatecontaining 0.2 M sodium chloride and dextran (3 mg/ml) may be dried onthe inside of the transparent nonporous surface of the viewing window.

(10 mg/ml) at the start of the assay. The blank site, Site D contains nosubstrate, phenolphthalein glucuronide. A blood sample is placed on bothSites D and E for analysis.

This screening system develops higher levels of color for low levels ofcholesterol. Since an excess of the substrate and enzyme are present,the rate of development of the color must be compared in order toestimate the assay. Thus, at and minutes after the start of the test,the pink red color of the test site should be compared with thestandards. Eventually the three standards and the test site will showthe same color development.

Other metabolites and substrates which may be determined utilizingsimilar procedures modified to accomodate spot techniques are: acetone,albumin, ascorbic acid, bilirubin, carotene, creatinine, creatine,glucose, pyruvic acid, urea nitrogen, porphyrin, cystine, hippuric acid,lactic acid, lactose, melanin, and pentose.

Manifestily minor changes and minor variations can be effected withoutdeparting from the spirit and scope of the invention as defined in andlimited solely by the appended claims.

We claim:

1. in a system for quantitating metabolite and enzyme substrateconcentrations in body fluids such as whole blood, urine, cerebrospinalfluid and the like:

A. a support;

B. means constituting a plurality of separate restricted test zone areasin a rigid array mounted on said support;

C. at least one of said areas including as a stacked array a pluralityof superposed test reagent impregnated members in a rigidly confinedcolumn which is adapted for placement of a fluid test media thereon,said stacked array including in descending sequence:

i. a porous upper glass fiber disc;

ii. a porous lower glass fiber disc;

iii. a membrane filter disc;

iv. a porous glass fiber filter disc; and v. a transparent membrane;

D. said upperand lower glass fiber discs constituting filters to removeamorphous matter including white blood cells and to partially removered-blood cells to prevent such matter from subsequently clogging poresof said membrane filter disc;

E. said upper and lower glass fiber discs constituting dried reagentstorage reservoirs for elution therefrom by clear filtrate passingtherethrough;

F. said membrane filter disc constituting a unit for removal of redblood cells, platelets, soluble proteins and remaining particles from atest specimen;

G. said upper and lower porous glass fiber discs and said porous glassfiber filter disc constituting fluid volume control units and saidporous glass fiber filter disc additionally functioning to assist indrawing liquid through said membrane filter disc and to distributereacting liquid consisting of the eluted filtrate and reagents acrosssaid transparent membrane which constitutes a viewing window;

H. said support including:

i. upper and lower slides; ii. said slides mounting therebetween saidstacked array; and

iii. means securing said slides together in fixed relationship.

2. In a system for quantitating metabolite and enzyme substrateconcentrations in body fluids such as whole blood, urine, cerebrospinalfluid and the like:

A. a support;

B. means constituting a plurality of separate restricted test zone areasin a rigid array mounted on said support;

C. at least one of said areas including as a stacked array a pluralityof superposed test reagent impregnated members in a rigidly confinedcolumn which is adapted for placement of a fluid test media thereon,said stacked array including in descending sequence:

i. a porous upper glass fiber disc; ii. a porous lower glass fiber disc;iii. a membrane filter disc;

iv. a porous glass fiber filter disc; and v. a transparent membrane;

D. said upper and lower glass fiber discs constituting filters to removeamorphous matter including white blood cells and to partially remove redblood cells to prevent such matter from subsequently clogging pores ofsaid membrane filter disc;

E. said upper and lower glass fiber discs constituting dried reagentstorage reservoirs for elution therefrom by clear filtrate passingtherethrough;

F. said membrane filter disc constituting a unit for removal of redblood cells, platelets, soluble proteins and remaining particles from atest specimen;

G. said upper and lower porous glass fiber discs and said porous glassfiber filter disc constituting fluid volume control units and saidporous glass fiber filter disc additionally functioning to assist indrawing liquid through said membrane filter disc and to distributereacting liquid consisting of the eluted filtrate and reagents acrosssaid transparent membrane which constitutes a viewing window;

H. said support including:

i. upper and lower slides;

ii. a top retaining ring mounted on the top of said upper slide;

iii. a lower retaining ring mounted on the top of said lower slide;

iv. the stacked array mounted between said upper and lower retainingrings;

v. means securing together corners of said slides;

and

vi. spring clamps operatively engaged over and covering the edges ofsaid slides for maintaining effective contact among the membersconstituting the stacked array confined between said retaining rings.

3. In a system as claimed in claim 2, calibrated and shaded color testcomparison means associated with the rigid array for visual comparisonwith color developed in a test zone area.

4. In a system as claimed in claim 3, said comparison means including amovable color shaded slide and a coacting calibrated scale.

5. In a system as claimed in claim 3, said comparison means including arevolvable color shaded disc and a coacting calibrated scale.

6. in a system as claimed in claim 3, said comparison means including afixed color shaded strip and a coacting calibrated scale.

7. In a system as claimed in claim 2, said upper and lower glass fiberdiscs impregnated with a blood uric acid test composition comprising drybovine serum albumin, glycine-borate buffer, uricase and the uppersurface of the transparent membrane having thereon a dry deposit ofO-Dianisidine Hydrochloride-Peroxidase mixture and dextran.

8. In a system as claimed in claim 2, said upper and lower glass fiberdiscs and said porous glass fiber filter disc impregnated with atestosterone and androsterone test composition comprising dry humanserum albumin, Nicotinamide adenine dinucleotide HydroxysteriodDehydrogenase, N-methyl phenazonium metho sulfate and the upper surfaceof the transparent membrane having thereon a dry deposit of a solutionof Nitro Blue tetrazolium and dextran.

9. In a system as claimed in claim 2, said discs impregnated with agalactose test composition comprising dry human serum albumin andGalactose oxidase, and a dry deposit of a solution of O-dianisidine,Peroxidase and Dextran on the upper surface of the transparent membrane.

10. In a system as claimed in claim 2, said discs for cholesterolscreening including on the upper and lower glass fiber discs dry serumalbumin, a dry deposit of an enzyme solution containing B-glucuronidasein acetate buffer, and the porous glass fiber filter disc beingimpregnated with a dry deposit of a substrate solution containingphenolphthalein glucuronide in acetate buffer and the upper surface ofthe transparent membrane having thereon a precipitated base reactible toobtain anionic red color from released phenolphthal- 11. In a system asclaimed in claim 2, said glass fiber filter disc having a freeze drieddeposit of a first selected reagent thereon and said upper and lowerglass fiber discs each having a freeze dried deposit of a secondselected reagent required for a given test impregnated thereon.

2. In a system for quantitating metabolite and enzyme substrateconcentrations in body fluids such as whole blood, urine, cerebrospinalfluid and the like: A. a support; B. means constituting a plurality ofseparate restricted test zone areas in a rigid array mounted on saidsupport; C. at least one of said areas including as a stacked array aplurality of superposed test reagent impregnated members in a rigidlyconfined column which is adapted for placement of a fluid test mediathereon, said stacked array including in descending sequence: i. aporous upper glass fiber disc; ii. a porous lower glass fiber disc; iii.a membrane filter disc; iv. a porous glass fiber filter disc; and v. atransparent membrane; D. said upper and lower glass fiber discsconstituting filters to remove amorphous matter including white bloodcells and to partially remove red blood cells to prevent such matterfrom subsequently clogging pores of said membrane filter disc; E. saidupper and lower glass fiber discs constituting dried reagent storagereservoirs for elution therefrom by cleAr filtrate passing therethrough;F. said membrane filter disc constituting a unit for removal of redblood cells, platelets, soluble proteins and remaining particles from atest specimen; G. said upper and lower porous glass fiber discs and saidporous glass fiber filter disc constituting fluid volume control unitsand said porous glass fiber filter disc additionally functioning toassist in drawing liquid through said membrane filter disc and todistribute reacting liquid consisting of the eluted filtrate andreagents across said transparent membrane which constitutes a viewingwindow; H. said support including: i. upper and lower slides; ii. a topretaining ring mounted on the top of said upper slide; iii. a lowerretaining ring mounted on the top of said lower slide; iv. the stackedarray mounted between said upper and lower retaining rings; v. meanssecuring together corners of said slides; and vi. spring clampsoperatively engaged over and covering the edges of said slides formaintaining effective contact among the members constituting the stackedarray confined between said retaining rings.
 3. In a system as claimedin claim 2, calibrated and shaded color test comparison means associatedwith the rigid array for visual comparison with color developed in atest zone area.
 4. In a system as claimed in claim 3, said comparisonmeans including a movable color shaded slide and a coacting calibratedscale.
 5. In a system as claimed in claim 3, said comparison meansincluding a revolvable color shaded disc and a coacting calibratedscale.
 6. In a system as claimed in claim 3, said comparison meansincluding a fixed color shaded strip and a coacting calibrated scale. 7.In a system as claimed in claim 2, said upper and lower glass fiberdiscs impregnated with a blood uric acid test composition comprising drybovine serum albumin, glycine-borate buffer, uricase and the uppersurface of the transparent membrane having thereon a dry deposit ofO-Dianisidine Hydrochloride-Peroxidase mixture and dextran.
 8. In asystem as claimed in claim 2, said upper and lower glass fiber discs andsaid porous glass fiber filter disc impregnated with a testosterone andandrosterone test composition comprising dry human serum albumin,Nicotinamide adenine dinucleotide Hydroxysteriod Dehydrogenase, N-methylphenazonium metho sulfate and the upper surface of the transparentmembrane having thereon a dry deposit of a solution of Nitro Bluetetrazolium and dextran.
 9. In a system as claimed in claim 2, saiddiscs impregnated with a galactose test composition comprising dry humanserum albumin and Galactose oxidase, and a dry deposit of a solution ofO-dianisidine, Peroxidase and Dextran on the upper surface of thetransparent membrane.
 10. In a system as claimed in claim 2, said discsfor cholesterol screening including on the upper and lower glass fiberdiscs dry serum albumin, a dry deposit of an enzyme solution containingB-glucuronidase in acetate buffer, and the porous glass fiber filterdisc being impregnated with a dry deposit of a substrate solutioncontaining phenolphthalein glucuronide in acetate buffer and the uppersurface of the transparent membrane having thereon a precipitated basereactible to obtain anionic red color from released phenolphthalein. 11.In a system as claimed in claim 2, said glass fiber filter disc having afreeze dried deposit of a first selected reagent thereon and said upperand lower glass fiber discs each having a freeze dried deposit of asecond selected reagent required for a given test impregnated thereon.